Our section has a major effort devoted to understanding the molecular and cellular basis of stuttering, a common but poorly understood speech disorder. Stuttering has long been known to have a genetic component, and we have previously identified mutations in the GNPTAB, GNPTG and NAGPA genes that are associated with stuttering in populations worldwide. These results indicate that non-syndromic stuttering can be associated with partial loss-of-finction mutations in lysosomal targeting and transport, a cellular function that is well-studied in health and disease. More recently we have identified mutations in the AP4E1 gene that cause stuttering. AP4E1 encodes a cellular component that serves to direct movement of vesicles within the cell, including those destined to go to the lysosome. The product of the AP4E1 genes recognizes and directs the trafficking of the product of the NAGPA gene in particular, thus tying this finding to our previous gene findings, and highlighting the importance of this cellular process in the genesis of stuttering. During the past year we have more closely examined the contribution of mutations in these four gene to stuttering worldwide. We found that a mutation in one of these four genes in 20% of unrelated individuals with familial persistent stuttering. However approximately 8% of individuals in existing genomic databases carry a mutation in one of these genes, so the true contribution of mutations in these genes to stuttering may be well under 20% of cases. We note that individuals in the genomic databases are all of unknown speech phenotype, and some of these individuals are certain to stutter, or to have stuttered in childhood. Evaluation of a group of matched controls who were determined to be normal following a detailed neurological history and examination showed a mutation in one of these genes in less than 2% of individuals. Thus the true contribution of mutations in these four genes to stuttering overall may well be close to 20%. However this indicates a large fraction of presumably genetic cases of stuttering have a causative gene that is not yet identified. We know from previous genetic linkage studies in our lab that other stuttering genes exist, and that additional genes that can cause stuttering reside on chromosomes 2, 3, 10, 14, and 16. Efforts are underway to identify these genes, with the goal of broadening our knowledge of the underlying causes of stuttering at the molecular and cellular level. Another goal of our current research is to determine how the cell metabolic defects caused by mutations in these genes lead to stuttering without any other discernible symptoms. To facilitate these and other studies, we have been working to develop a mouse model of human stuttering. This is being done by creating so-called knock-in strains of mice that carry the mutations identified in humans who stutter. These experiments require a detailed acoustical analysis of mouse vocalization, which is largely ultrasonic in nature. In these experiments, we are working with Drs. Terra Barnes and Tim Holy at Washington University in St. Louis. Our studies have demonstrated that the presence of human stuttering mutations causes reproducible alterations in mouse vocalizations, and that these alterations show parallels with the alterations present in the speech of individuals who stutter. We are currently using these mice to identify specific anatomic or cellular alterations that accompany this vocalization deficit, using three different approaches. The first approach is staining brain tissue with antibodies that recognize surface molecules on specific cell types in the brain, and comparing these cells in mutant animals with those in their wild-type littermates. The second approach is to use additional mouse genome engineering techniques to express stuttering mutations within specific neuronal cell types and cell lineages within the brain using neuronal cre- driver lines. Testing the ultrasonic vocalizations of these mice are expected to provide an independent but parallel approach to identify specifically which cell types within the brain mediate these alterations in vocalization. The third approach looks are larger scale brain structures in these mice using MRI and DTI. This is being done in collaboration with the NIH Mouse Imagining Facility using a 7T scanner. Our studies of taste perception are focused on the role of genetic differences in taste perception in tobacco use, particularly the use of mentholated cigarettes, which are disproportionately used by African Americans. Our goal is to determine whether this disproportionate use is associated with genetic differences, specific to African Americans, in genes that encode taste perception machinery. This study is being done in collaboration with the University of Texas Southwestern Medical Center, using the well-characterized Dallas Heart Study population. In the past year, we have also entered into a collaboration with Dr. Thomas Kirchner at New York University and Dr. Ray Niaura at the Schroeder Center for Tobacco Research, who together have provided >1700 DNA samples from individuals with known smoking phenotypes. In the past year, whole exome genotyping and sequencing have identified a surprising association with a coding variant in the MRGPRX4 gene. This variant exists only in populations of African origins. While the function of MRGPRX4 is poorly understood, it is expressed primarily in sensory neurons, where it appears to be involved in nociception. These findings have been submitted for publication and are currently under review.